Single-Cell PCR Analysis of T Helper Cells in Human Lymph Node Germinal Centers

Abstract

The T helper cell population of human lymph node germinal centers (GCs) was analyzed for clonality and signs of antigen selection. Frozen sections of lymph node biopsies taken from three different individuals were used to micromanipulate single T cells from one particular GC for each of the specimens. T cell receptor (TCR) beta gene rearrangements were amplified from these single cells and directly sequenced. Although only unique rearrangements were amplified from T cells of GC2 and GC3, 11 of 28 potentially functional rearrangements amplified from GC1 originated from four different clones. In all three GCs, TCR gene rearrangements neither showed obvious biases in gene segment usage nor similarities in complementarity determining region 3 amino acid sequence. Thus, it appears that T lymphocytes in human GCs usually represent a diverse population of cells. Sequence analysis of V region genes did not provide evidence that in the human the process of somatic hypermutation acts on the TCRbeta loci. For one of the GCs (GC3), immunoglobulin heavy chain (IgH) gene rearrangements were amplified and sequenced from single micromanipulated GC B cells. The detection of clonal expansions accounting for more than half of the sampled B cells in addition to ongoing somatic hypermutation of Ig V region genes suggested that GC3 was a fully developed GC.